1. Field of the Invention
The invention relates to alleles of the thrA gene of Enterobacteriaceae coding for desensitized aspartokinase I-homoserine dehydrogenase I enzymes and methods for the fermentative production of L-threonine using bacteria of the family Enterobacteriaceae containing these alleles.
2. Background Information
L-threonine is used in human medicine, in the pharmaceutical industry, in the food industry and particularly in animal nutrition.
It is known that L-threonine is produced by fermenting strains of Enterobacteriaceae, especially Escherichia coli (E. coli) and Serratia marcescens. Because of its great importance, constant work is being carried out to improve production methods. Method improvements can relate to fermentation measures such as, for example, agitation and supplying with oxygen or the composition of the nutrient media such as, for example, the concentration of sugar during the fermentation or can concern the workup to product form by, for example, ion exchange chromatography or concern the intrinsic performance properties of the microorganism itself.
Methods of mutagenesis, selection and mutant selection are used to improve the performance properties of these microorganisms. In this manner, strains are obtained that are resistant to antimetabolites such as, for example, the threonine analogue α-amino-β-hydroxyvaleric acid (AHV) or are auxotrophic for metabolites important in regulation and that produce L-threonine.
For several years methods of recombinant DNA technology for the improvement of strains of the family Enterobacteriaceae producing amino acids have also been used to amplify individual amino-acid biosynthesis genes and examine the effect on production.
Two important reaction steps in the biosynthesis of L-threonine are the aspartokinase reaction and the homoserine dehydrogenase reaction. Aspartokinase catalyzes the conversion of L-asparaginic acid to aspartyl phosphoric acid. Homoserine dehydrogenase catalyzes the conversion of asparaginic acid semialdehyde to homoserine.
There are three isoenymes in Escherichia coli K-12 and Serratia marcescens which catalyze these reactions: Aspartokinase III encoded by the lysC gene, the bifunctional enzyme aspartokinase II-homoserine dehydrogenase II encoded by the metL gene, and the bifunctional enzyme aspartokinase I-homoserine dehydrogenase I encoded by the thrA gene. The enzyme aspartokinase I-homoserine dehydrogenase I in particular is responsible for the biosynthesis of L threonine. Summarizing presentations for this are found, for example, in the textbook of Frederick C. Neidhard “Escherichia coli and Salmonella, Cellular and Molecular Biology” (2nd edition, ASM press, USA, Washington D.C., 1996).
The nucleotide sequence of the thrA gene of Escherichia coli is known and available under the accession number AE000111 in the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA). Mutants were isolated by Saint-Girons and Margarita (Journal of Bacteriology 124, 1137-1141 (1975)) with the aid of the threonine analogue α-amino-β-hydroxyvaleric acid (AHV) which mutants were able to excrete L-threonine. The mutation site was, according to the authors, in the thrA gene, and the enzyme aspartokinase I-homoserine dehydrogenase I was resistant to inhibition by threonine.
The nucleotide sequence of the thrA gene of Serratia marcescens is known and available under the accession number X60821 in the NCBI. Mutants were isolated from Serratia marcescens Sr41 which mutants produce L-threonine and have mutations in the thrA gene. These thrA alleles were sequenced and are described in Omori et al. (Journal of Bacteriology 175, 785-794 (1993)). The proteins encoded by these thrA alleles have amino-acid exchanges at position 330 and position 352 and position 479 of the aspartokinase I-homoserine dehydrogenase I protein.